Iranian Journal of Pharmaceutical Research (20 14), 13 (supplement): 125-132
Received: April 2013
Accepted: September 2013
Copyright © 2014 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
The Effect of Paxilline on Early Alterations of Electrophysiological
Properties of Dentate Gyrus Granule Cells in Pilocarpine-Treated Rats
Nasrin MehranfarcH, Hamid Gholamipour-Badie 6 c , Fereshteh Motamedi 6 , Mahyar JanahmadP >c
and Nima Naderi a * 6 *
a Department of Pharmacology and Toxicology, School of Pharmacy, Shahid Beheshti
University of Medical Sciences, Tehran, Iran. h ^N euro science Research Center, Shahid Beheshti
University of Medical Sciences, Tehran, Iran, department of Physiology, Faculty of Medicine,
Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Abstract
The dentate gyrus of hippocampus has long been considered as a focal point for studies
on mechanisms responsible for the development of temporal lobe epilepsy (TLE). Change in
intrinsic properties of dentate gyrus granule cells (GCs) has been considered as an important
factor responsible in temporal lobe seizures. In this study we evaluated the intrinsic properties
of GCs, during acute phase of seizure (24 h after i.p. injection of pilocarpine) compared to sham
group using whole cell patch-clamp recordings. Our results showed a significant increase in the
number of action potentials (APs) after applying depolarizing currents of 200 pA (p < 0.01)
and 250pA (p < 0.05) compared to sham group. The evaluation of AP properties revealed a
decrease in half-width of AP in GCs of seizure group (1.27 ± 0.03 ms) compared to sham group
(1.60 ± 0.11). Moreover, addition of BAPTA to pipette solution prevented changes in AP half-
width in seizure group (1.71 ± 0.11 ms) compared to sham group (1.91 ± 0.08 ms). In contrast,
an increase in the amplitude of fast afterhyperpolarization was observed in GCs of seizure
group (-11.68 ± 0.72 mV) compared to sham group (-8.28 ± 0.59 mV). Also, GCs of seizure
group showed a significant increase in both firing rate and instantaneous firing frequency at
depolarizing currents of 200 pA (P < 0.01) and 250 pA (P < 0.05) compared to sham group.
The changes in electrophysiological properties of GCs were attenuated after bath application
of paxilline suggesting possible involvement of large conductance Ca 2+ - activated K + channel
(BK channel). Our results suggested the possible involvement of certain potassium channels
in early changes of intrinsic properties of GCs which eventually facilitate TLE development.
Keywords: Paxilline; Dentate gyrus; Granule cells; Epilepsy.
Introduction
Temporal lobe epilepsy (TLE) is the most
common form of acquired epilepsy in adult and
is often resistant to antiepileptic drug-therapy
(1). In animal models, TLE is a situation that
Corresponding author:
E-mail: naderi.nima@gmail.com
is induced after injection of pilocarpine (2),
kainic acid (3), or electrical stimulation of a
specific site of brain (4,5) and is associated with
recurrent spontaneous seizures. Pilocarpine is a
potent Ml muscarinic agonist and its systemic
injection to rodents induces behavioral and
electrophysiological changes in three distinct
phases including acute phase which is caused
by an initial brain insult and lasts 24 h, a latent
MehranfardN etal. I IJPR (2014), 13 (supplement): 125-132
phase that is a relatively seizure-free period and
lasts between 4-44 days and a chronic phase in
which spontaneous recurrent seizure occurs
(6) . After a brain insult, a cascade of brain
reorganization events termed epileptogenesis is
induced which lead to changes in brain excitability
and occurrence of spontaneous recurrent seizures
(7) . Dentate gyrus granule cells (GCs) has long
been recognized as a focal point for studies on
mechanisms responsible for epileptogenesis. In
recent years, much attention has been focused
on changes in the intrinsic properties of neurons
especially the role of K + channels as a possible
mechanism in epileptogenesis and generation
of hippocampal epilepsy. For instance, Brenner
and coworkers in 2005 reported that p4 subunit
of large conductance Ca 2+ activated K + channels
(BK channels) reduces excitability of dentate
gyrus GCs preventing temporal lobe seizures (8).
Despite many studies on hippocampal
seizure, there is little information regarding
early events that initiate epileptogenesis in
dentate gyrus. Identification of early alterations
in electrophysiological properties of GCs would
enable us to know how these changes might
contribute to the epilepsy. In this study, we
evaluated changes in the intrinsic properties of
GCs during acute phase of pilocarpine-induced
seizure using whole cell patch clamp recordings.
Experimental
Animals
Male wistar rats weighting 150-200 g
(Pasteur Institute, Tehran, Iran) were used in this
study. The animals were housed in colony cages
(5 rats per cage) with free access to food and tap
water under standardized housing conditions
with a 12 h light-dark cycle (lights on at 7:00
a.m.) and temperature-controlled (22 ± 1 °C)
environment. All procedures were in accordance
with the National Institute of Health Guide for
the Care and Use of Laboratory Animals (NIH
Publications No. 80-23, revised 1996) and were
approved by the local Research and Medical
Ethics Committee.
Drugs
Rats were treated with pilocarpine (350
mg/Kg, ip\ Sigma-Aldrich Co. St Louis,
USA) 20 min after methyl scopolamine (5
mg/Kg, S.C; Sigma-Aldrich Co. St Louis, USA)
administration in order to reduce the peripheral
effects of pilocarpine. Diazepam (4 mg/kg,
ip; Sigma-Aldrich Co. St Louis, USA) was
administered after 3 h to stop status epilepticus
(SE). Only motor seizures of grade 3 or greater
on the Racine scale (9) were scored.
Patch-clamp recordings in hippocampal
slices
Slice preparation
Twenty-four hours after seizure induction,
rats were anaesthetized with ether and then
decapitated. The brains were quickly removed
and chilled in ice-cold slicing solution
containing (in mM): 125 NaCl, 2.8 KC1, 1
CaCl 2 , 1 MgCl 2 , 2 MgS0 4 , 1.25 NaH 2 P0 4 , 26
NaHC0 3 , 10 D-glucose and set to pH of 7.4
(with 95% oxygen and 5% carbon dioxide);
the osmolarity was adjusted to 305 mOsm by
addition of sucrose to the solution. The brain
transverse slices containing hippocampal area
were cut into 350-400 \im using a vibroslicer
(752 M, Campden Instruments Ltd, UK). The
slices were then incubated in ACSF containing
(in mM): 124 NaCl, 2.8 KC1, 2 CaCl 2 , 2 MgS0 4
,1.25 NaH 2 P0 4 , 26 NaHC0 3 , and 10 D-glucose
at pH 7.4, the osmolarity of 295 mOsm and
temperature of 32-35 °C for lh and stored at
22-24 °C (room temperature) before being
transferred to the recording chamber.
Electrophysiology
The slices were transferred to a submerged
recording chamber and were continuously
perfused with ACSF (1-2 mL/min) at room
temperature. Dentate gyrus GCs were visualized
by infrared videoimaging (Hmamatsu, ORSA,
Japan) with a 40x water immersion objective.
Recordings were made using glass electrodes
pulled with a two-stage vertical puller (PC 10,
Narishige, Japan) from borosilicate glass
capillary (1.2 mm O.D., 0.95 mm I.D.). The
pipettes had a resistance of 3-6 MQ and filled
with intracellular solution containing (in mM)
135 potassium methylsulfate (KMeS0 4 ), 10
KC1, 10 HEPES, 1 MgCl 2 , 2 Na 2 ATP, and 0.4
Na 2 GTP. The pH of internal solution was adjusted
to 7.3 by KOH, and the osmolarity was set to
126
The Effect of Paxilline on Early Alterations of Electrophysiological
295 mOsm. Whole-cell patch-clamp recordings
were made from dentate gyrus GCs using
Multiclamp 700B amplifier (Axon Instruments,
Foster City, CA) equipped with Digidata 1320
A/D converter (Axon Instruments, Foster City,
CA). Recordings were only obtained when seals
of more than 1GQ resistance were established.
The function of test seal was frequently checked
during the experiment to ensure that the seal is
stable. Access resistance was less than 20 MQ
and less than 20% change during recordings
was acceptable in order to include the recording
for further analysis. In addition, only cells
with resting membrane potential (RMP) more
hyperpolarized than -70 mV, input resistance
(R. n ) >200 MQ, and obvious overshoot of action
potential (AP) were included in analysis. In
certain recordings, paxilline (1 |LiM; Sigma-
Aldrich Co. St Louis, USA) was included in
bath solution (10), and 1,2-Bis (2-amino-5-
bromophenoxy) ethane-A^A^TV'-tetraacetic
acid (BAPTA; 10 mM) was applied in the pipette
solution . Electrophysiological recordings
were sampled at 10 kHz, filtered at 5 kHz
and stored for offline analysis. To investigate
the electrophysiological properties of GCs in
current clamp mode, trains of APs were elicited
by applying depolarizing currents (50-250pA;
1000 ms) while the cell was hold at -75 mV. AP
characteristics were measured based on the first
AP elicited by the depolarizing current of 200pA
(11). Passive and active electrophysiological
parameters including RMP, R n , the number
of APs, fast after-hyperpolarization (fAHP)
amplitude, AP duration at half-width, decay
time, instantaneous firing frequency (IFF) were
measured. R n was defined by the steepest slope of
the I-V curve based on steady-state responses to
hyperpolarizing current pulses (50-200 pA, 300
ms). The AHP amplitude was measured from the
level of RMP to the peak of the hyperpolarization.
AP half- width was measured before and 15 min
after bath application of paxilline to ensure
that maximum effects were obtained. IFF was
measured as 1 /interval between the first and the
second AP.
Statistical analysis
Data were shown as mean ± S.E.M. The
Student) s unpaired t-test, one-way ANOVA and
two-way ANOVA followed by Bonferroni's
post-test were used as appropriated. A p-value
less than 0.05 were considered statistically
significant.
Results
The passive membrane properties of GCs,
such as RMP and R in did not affect by pilocarpine -
induced seizure nor by Paxilline application
(data not shown).
Paxilline reversed hyperexcitability of
dentate gyrus GCs in acute phase of pilocarpine-
induced seizure
The excitability of GCs during acute phase
of seizure was evaluated by measuring the
number of APs elicited by depolarizing current
pulses ranging from 50 to 250 pA. Figure 1A
showed traces of APs. Injection of depolarizing
currents into granule cells significantly increased
the number of action potentials in acute seizure
group compared to sham group [F (1 , 72) = 23 .06,
p < 0.0001; Figure IB]. Further analysis using
Bonferroni's post-test revealed a significant
increase in number of APs at 200 (p < 0.01) and
250 pA (p < 0.05) compared to sham group. Bath
application of paxilline significantly attenuated
seizure-induced increase in APs at 200pA (p <
0.05) and 250 pA (p < 0.01) compared to seizure
group.
Changes in fAHP and AP half-width during
acute phase of pilocarpine-induced seizure were
reversed by paxilline
In order to further illustrate the mechanisms
involved in regulation of GCs firing rate, fAHP
and half-width of the first AP were measured
after injection of the depolarizing current pulse
of 200pA. Figure 2A shows a representative
trace of the firs AP in sham, acute seizure, and
seizure -paxilline group. As shown in Figure
2B, the fAHP of the first AP was significantly
changed in different groups [F (3, 25) = 4.48; p
= 0.012]. Further analysis revealed a significant
increase (p < 0.01) in fAHP of cells in seizure
group (-11.67 ± 0.72 mV; N = 8) compared to
sham group (-8.28 ± 0.59 mV; N = 8). Bath
application of paxilline, significantly reversed
seizure-induced increase in fAHP amplitude
127
MehranfardN etal. I IJPR (2014), 13 (supplement): 125-132
Sham
Acute seizure
Acule-paxiHline
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Sham-Paxilline
Acute Seizure
Acute Seizure-Paxilline
JL
100
150 200
Current injection(pA)
250
Figure 1. Alteration in the firing rate of dentate gyrus GCs recorded 24 h after pilocarpine-induced seizure. (A) Representative traces
show the differences in firing rate of the GCs in response to 1000 ms depolarizing pulses from 50 pA to 250 pA in sham (above), acute
seizure (middle) and acute seizure + paxilline (down) groups. (B) Depolarizing current injection ranging from 50 pA to 250 pA increased
the number of APs in seizure group compared to sham group. The application of paxilline (1 uM) reversed pilocarpine-induced increase
in APs. Data were shown as mean+SEM (N = 10).
*p<0.05, **p<0.01 significant difference compared to sham group.
+p < 0.05, ++p<0.01 significant difference compared to acute seizure group.
(p < 0.01) toward sham group (-9.26 ± 0.42
mV, N = 8). Moreover, as shown in Figure 2C,
a significant change was observed in the half-
width of AP in different groups [F (3, 23) =
6.605; p = 0.002]. Further analysis revealed a
significant decrease (p < 0.01) in the half- width
of the first AP in seizure group (1.27 ± 0.03 ms; N
= 7) compared to sham group (1.60 ±0.11 ms; N
= 8). Decrease in AP half- width of seizure group
could be due to decrease in decay time of AP
(Figure 2D), suggesting the role of K + channels
in reduction of half-width. Bath application
of paxilline, significantly reversed (p < 0.01)
seizure-induced changes in the half-width of AP
toward sham group (1.69 ± 0.08 ms; N = 8).
Application of BAPTA increased half width
to normal values
The Ca 2+ -activated K + channels are activated
by both membrane depolarization and increase
in intracellular Ca 2+ concentration [Ca 2+ ].
(12,13). In order to examine the role of [Ca 2+ ]
128
The Effect
of Paxilline on Early Alterations of Electrophysiological
Figure 2. Pilocarpine-induced changes in AHP amplitude and AP half-width were reversed by bath application of paxilline.
(A) Representative trace of 1st AP of sham, acute seizure and acute seizure + paxilline groups during a train of AP evoked by 200 pA
current injection for 1000 ms. (B) fAHP amplitude was measured from the level of RMP to the peak of the hyperpolarization. fAHP
amplitude significantly increased during acute phase of pilocarpine-induced seizure compared to sham group. Paxilline decreased fAHP
amplitude to the sham values. (C) AP half- width significantly decreased during acute phase of seizure. Paxilline increased AP half- width
to normal values and reversed the effect of seizure on AP half- width. (D) The decay time of AP was increased in pilocarpine-induced
seizure group and was reversed after bath application of paxilline.
*p<0.05, **p<0.01 significant difference compared to sham group.
++p<0.01, +++p<0.001 significant difference compared to acute seizure group.
##p<0.01 significant difference compared to sham + paxilline group.
. in firing rate of GCs, the Ca 2+ chelator BAPTA
(10 mM) was added to the internal solution from
both sham and acute seizure group. Application
of BAPTA prevented change in AP half- width in
seizure group (1.71 ±0.11 ms; N = 5) compared
to that of BAPTA-sham cells (1.91 ± 0.08 ms;
N = 5), suggesting a role of intracellular Ca 2+ in
firing frequency (Figure 3).
Increase in IFF after seizure is reversed by
paxilline application
As shown in Figure 4, IFF was significantly
enhanced after seizure [F (2, 39) = 15.94, P <
0.0001]. Further analysis using Bonferroni's
post-test revealed a significant increase in IFF
at 200 pA (P < 0.01) and 250 pA (P < 0.05)
129
MehranfardN etal. I IJPR (2014), 13 (supplement): 125-132
Figure 3. Effect of BAPTA on AP half-width. Application of
BAPTA prevented change in AP half-width in seizure group
compared to that of BAPTA-sham cells. Data were shown as
mean + SEM (N = 5).
compared to sham group. Bath application of
paxilline significantly reduced IFF to sham
values at 150 pA (P < 0.05), at 200 pA (P < 0.01),
and 250 pA (P < 0.05), compared to those of
sham group, suggesting the possible role of BK
channel in enhancement of the firing rate.
Discussion
There are several documents that show
the role of dentate gyrus in epileptogenesis
(14). Although seizure-induced changes in
hippocampal neurons have been studied
extensively, the mechanisms that initiate
epileptogenesis have not been fully established.
In this study, we focused on intrinsic
membrane properties of granule cells 24 h
after seizure induction and demonstrated that
pilocarpine-induced seizures altered intrinsic
membrane properties of the dentate gyrus GCs.
Pilocarpine caused a significant increase in the
firing frequency, the fAHP amplitude, and the
IFF while caused a significant reduction in AP
half- width and decay time in GCs during acute
phase of seizure. Previous studies reported
that pilocarpine-induced seizure induces
hyperexcitability of hippocampal cells during
early stages of epileptogenesis. Using field
potential recording, a hyperexcitability, as
a transient increase of the input and output
field responses, has been shown during the
latent period of epileptic animals which may
participate in development of epilepsy (15). In
other study, patch-clamp recording from GCs
in dentate gyrus revealed that the stimulation
of perforant pathway produce hyperexcitability
of GCs as an increase in the number of action
potentials (16). In our study, for the first
time, a significant change in certain intrinsic
properties of GCs was reported which could
result in hyperexcitability of these cells. Also,
our results showed that bath application of
paxilline attenuated the increase in firing rate
of GCs to normal values and reversed the
effects of pilocarpine on fAHP amplitude, AP
half-width, decay time and IFF, suggesting the
role of K + channels, including BK channels, in
hyperexcitability of GCs during acute phase of
TLE. The BK channels are widely expressed
in CNS and are gated both by voltage and by
intracellular Ca 2+ ions. These channels not only
contribute to action potential repolarization
and shape the fAHP (17,18), but can also affect
neuronal firing patterns (19,20). A relation
between seizure and gain-of-function of BK
channel has been associated with high firing
rate of neocortical neurons with an increase
in the AHP amplitude and a decrease in AP
half- width (21). Moreover, a gain- of-function
of BK channel activity in genetic epilepsy
both in human and mice have been associated
with recurrent seizures (22,8). The changes
in neuronal excitability has been shown in
other conditions related to synaptic plasticity,
such as learning, where it could be modulated
by changing the amplitude of AHP (23).
BK channels are one of the most prominent
ion channels which have been shown to be
involved in the generation of the fAHP (24).
The mechanism by which BK channel activity
increases the firing rate might contribute to a
faster repolarization and a more deinactivation
of Na + channels that occurs during the fAHP,
increasing Na + channel availability and this
resulted in firing with short latencies (19). The
observed changes in the intrinsic properties
of GCs is likely attributed to [Ca 2+ ]i, as the
elimination of intracellular Ca 2+ using BAPTA
130
The Effect of Paxilline on Early Alterations of Electrophysiological
80-
60-
x
40-
LL
20-
Sham
Acute Seizure
Acute Seizure-Paxilline
150 200 250
Current injection (pA)
Figure 4. Seizure-induced increase in instantaneous firing
frequency (IFF) was reversed by paxilline application. IFF was
measured as the reciprocal of interval between the first and the
second AP during 200 pA current injection. IFF significantly
increased during acute phase of pilocarpine-induced seizure.
After bath application of paxilline, IFF returned to sham levels.
Data were shown as mean ± SEM (N = 6 in each group).
*p<0.05, **p<0.01 significant difference compared to sham
group.
+p<0.05, ++p<0.01 significant difference compared to acute
seizure group.
reversed the decreased half-width of APs
during acute phase of TLE. Consistent with our
results, recent studies have shown an increase
of [Ca 2+ ]i in rat hippocampal CA1 neurons
during the acute phase of pilocarpine model
of seizure (25,26) which could induce gain-of-
function of BK channels.
In conclusion, these results suggest that
pilocarpine-induced hyperexcitability in dentate
gyrus GCs during acute phase could result from
alterations in the intrinsic properties of the cells,
particularly those related to potassium channels
activation which could give rise to an increase in
the firing rate of GCs. Considering the possible
role of BK channel activity in early stages of
epileptogenesis, the blockade of these channels
might have a potential therapeutic effect in
prevention of synaptic plasticity required for
recurrent seizure occurrence.
Acknowledgment
This work is part of Ph.D. dissertation of N.
Mehranfard at Shahid Beheshti University of
Medical Sciences.
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